The absorption and utilization of phosphate by plants mainly depend on phosphorus transporters, among which the low-affinity phosphorus transporters encoded by the PHT2 family are mainly responsible for the absorption, transport and reuse of phosphate by plants under normal phosphorus supply conditions. In order to explore the role and expression mode of low-affinity phosphorus transporter gene NtPHT2;1 in tobacco phosphate transport, this study cloned NtPHT2;1 using the cDNA of common tobacco K326 as a template, and conducted bioinformatics analysis of the gene and subcellular localization of the protein. The expression patterns of this gene under abiotic stress such as low phosphorus were analyzed by fluorescence quantitative PCR. The results showed that: (1) The total length of NtPHT2;1 gene is 1 764 bp and encodes 587 amino acids. (2) The results of subcellular localization showed that NtPHT2;1 protein was localized on chloroplasts. (3) Homology analysis showed that the homology between NtPHT2;1 protein and CaPHT2;1 protein reached 91.00%. (4) Promoter analysis showed that NtPHT2;1 promoter contains cis-acting elements involved in the regulation of plant aging and stress. (5) Tissue expression pattern analysis showed that the expression level of NtPHT2;1 was the highest in leaves, and the expression level in new leaves was higher than that in old leaves. There was no significant difference in the expression of this gene under low phosphorus induction compared with normal conditions. (6) The expression patterns under different abiotic stresses showed that the expression of this gene decreased significantly under salt stress and drought stress. It is concluded that NtPHT2;1 gene is mainly responsible for phosphate transport and utilization under normal growth and development of tobacco plants.